Use the Trypsin digestion buffer to re-swell the gel slice after it is dehydrated using acetonitrile, then add an amount of Trypsin to the sample in a ratio range of 1:10-20 wt./wt. enzyme:substrate. For example, a protein band estimated to be 1 ug would be combined with 50 to 100 ng of Trypsin and incubated at 25-37°C overnight. We generally clean-up our in-gel reactions on C18 ZipTips before MS (MALDI-MS or Ion Trap MS/MS).
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